The NF-B family of transcription factors regulates diverse biological processes, including immune response, inflammation, cell growth and survival. NF-B activation involves two major pathways: the canonical and noncanonical pathways. Despite the extensive studies of the canonical pathway, the noncanonical pathway is still poorly understood. Recent studies, pioneered by the PIs group, suggest that activation of the noncanonical NF-B pathway involves degradation of an inhibitory protein, TRAF3, and stabilization of the NF-B-inducing kinase (NIK). However, how these signaling events are negatively controlled is still unknown. This knowledge is highly significant, since deregulated activation of the noncanonical NF-B pathway is associated with severe human diseases, including lymphoid disorders and cancer. The overall objective of this continuation application is to characterize the molecular mechanisms mediating the negative regulation of signal-induced noncanonical NF-B signaling. The proposed studies are based on highly innovative preliminary data from our laboratory. In particular, we have identified two novel signaling factors the deubiquitinase Otud7b and the protein kinase TBK1, with pivotal roles in the negative regulation of noncanonical NF-B signaling. Our data suggest that Otud7b and TBK1 may act in different signaling steps to prevent aberrant activation of noncanonical NF-B. Using newly generated Otud7b knockout (KO) mice and TBK1 conditional KO mice, we have obtained evidence suggesting an important role for Otud7b and TBK1 in regulating antibody responses and maintaining lymphoid homeostasis in the intestine, an organ with dynamic host-microbiota interactions. Our central hypothesis is that Otud7b and TBK1 control the magnitude of signal-induced noncanonical NF-B activation and, thereby, maintain normal immune homeostasis and responses. To address this hypothesis and accomplish our overall objective, we will (1) examine how the deubiquitinase Otud7b controls signal-induced TRAF3 degradation and noncanonical NF-B activation; (2) elucidate the mechanism by which TBK1 regulates the fate of NIK and the magnitude of noncanonical NF-B signaling; and (3) characterize the in vivo role of Otud7b and TBK1 in regulating noncanonical NF-B signaling and immune functions. The PIs laboratory pioneered the studies that led to the discovery of the noncanonical NF-B pathway as well as to the elucidation of the mechanism underlying the steady-state noncanonical NF-B regulation. We believe that the studies proposed in the current application will once again lead to a major advancement of the field, since they address the currently unknown mechanism that negatively controls signal-induced noncanonical NF-B activation. In addition to the conceptual innovation, the newly generated Otud7b KO mice and TBK1 conditional KO mice serve as highly innovative models for studying the pathophysiology of noncanonical NF-B signaling.